EN ISO 20976-1:2019

SLOVENSKI STANDARD
01-julij-2019
Mikrobiologija v prehranski verigi - Zahteve in smernice za vodenje izzivnega
preskusa pri kmetijskih pridelkih in živilskih proizvodih - 1. del: Izzivni preskus za
potencial rasti, čas prilagajanja in najvišjo stopnjo rasti (ISO 20976-1:2019)
Microbiology of the food chain - Requirements and guidelines for conducting challenge
tests of food and feed products - Part 1: Challenge tests to study growth potential, lag
time and maximum growth rate (ISO 20976-1:2019)
Mikrobiologie der Lebensmittelkette - Leitfaden zur Durchführung von Challengetests bei
Lebensmitteln und Futtermitteln - Teil 1: Challengetests zur Untersuchung von
Wachstumspotential, der Verzögerungszeit und maximaler Wachstumsrate (ISO 20976-
1:2019)
Microbiologie de la chaîne alimentaire - Exigences et lignes directrices pour la réalisation
des tests d'épreuve microbiologique - Partie 1: Tests de croissance pour étudier le
potentiel de croissance, le temps de latence et le taux de croissance maximal (ISO
20976-1:2019)
Ta slovenski standard je istoveten z: EN ISO 20976-1:2019
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 20976-1
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2019
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Requirements and
guidelines for conducting challenge tests of food and feed
products - Part 1: Challenge tests to study growth
potential, lag time and maximum growth rate (ISO 20976-
1:2019)
Microbiologie de la chaîne alimentaire - Exigences et Mikrobiologie der Lebensmittelkette - Leitfaden zur
lignes directrices pour la réalisation des tests Durchführung von Challengetests bei Lebensmitteln
d'épreuve microbiologique - Partie 1: Tests de und Futtermitteln - Teil 1: Challengetests zur
croissance pour étudier le potentiel de croissance, le Untersuchung von Wachstumspotential, der
temps de latence et le taux de croissance maximal (ISO Verzögerungszeit und maximaler Wachstumsrate (ISO
20976-1:2019) 20976-1:2019)
This European Standard was approved by CEN on 21 January 2019.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20976-1:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 20976-1:2019) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”
the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by October 2019, and conflicting national standards shall
be withdrawn at the latest by October 2019.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 20976-1:2019 has been approved by CEN as EN ISO 20976-1:2019 without any
modification.
INTERNATIONAL ISO
STANDARD 20976-1
First edition
2019-03
Microbiology of the food chain —
Requirements and guidelines for
conducting challenge tests of food and
feed products —
Part 1:
Challenge tests to study growth
potential, lag time and maximum
growth rate
Microbiologie de la chaîne alimentaire — Exigences et lignes
directrices pour la réalisation des tests d'épreuve microbiologique —
Partie 1: Tests de croissance pour étudier le potentiel de croissance, le
temps de latence et le taux de croissance maximal
Reference number
ISO 20976-1:2019(E)
©
ISO 2019
ISO 20976-1:2019(E)
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

ISO 20976-1:2019(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 4
4.1 General . 4
4.2 Estimation of the growth potential . 6
4.3 Estimation of the growth kinetics parameters (lag time and maximum growth rate) . 7
5 Apparatus . 7
6 Culture media and reagents . 7
7 Study design and sampling . 8
7.1 General . 8
7.2 Setting decision criteria for growth potential . 8
7.3 Number of batches and selection criteria . 8
7.4 Preparation of the test units . 8
7.5 Number of test units to be inoculated. 9
8 Selection of strains . 9
9 Preparation of the inoculum .10
9.1 General .10
9.2 Preparation of the vegetative cell suspensions .10
9.3 Preparation of the spore suspensions .10
10 Inoculation of the tests units .10
11 Controls .11
11.1 Food controls .11
11.2 Control units.11
12 Storage of the test units .12
12.1 General .12
12.2 Estimation of growth potential .12
12.3 Estimation of growth kinetics parameters (lag time and growth rate) .12
13 Analysis .12
14 Expression of the results .13
14.1 General .13
14.2 Growth potential (Δ) .13
14.3 Growth kinetics parameters (lag time and growth rate) .14
15 Test report .14
15.1 General .14
15.2 Aim of the study and type of challenge test .14
15.3 Experimental protocol .15
15.4 Sample analysis .15
15.5 Results .15
15.6 Conclusions .16
15.7 Reference documents .16
Annex A (informative) Inter-batch variability assessment based on pH and a .17
w
Annex B (normative) Minimum number of units to prepare for the challenge test study .18
Annex C (informative) Examples of protocols to prepare inocula .19
ISO 20976-1:2019(E)
Annex D (informative) Examples of how to estimate growth potential, lag time and
maximum growth rate from results of challenge tests .22
Annex E (informative) Use of simulation to assess a microbial population under different
temperature conditions .26
Bibliography .27
iv © ISO 2019 – All rights reserved

ISO 20976-1:2019(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso
.org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
A list of all the parts in the ISO 20976 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
ISO 20976-1:2019(E)
Introduction
Under the general principles of the Codex Alimentarius on food hygiene, it is the responsibility of food
business operators (FBOs) to control microbiological hazards in foods and to manage microbial risks.
[11]
Therefore, FBOs implement validated control measures within the hazard analysis and critical
control point (HACCP) system, and conduct studies in order to investigate compliance with the food
safety criteria throughout the food chain.
In the framework of microbial risk assessment (MRA), several complementary approaches are developed
to estimate risks posed by pathogens or spoilage microorganisms in the food chain. MRA is adopted by
regulators under the auspices of the international agency for setting food standards. Challenge testing
is one of the recognized approaches used to validate control measures within the HACCP system, as
well as to assess microbiological safety and quality of food, food production processes, food storage
conditions and food preparation recommendations for consumers.
This document provides technical rules, calculations and approaches to investigate the ability of
inoculated microorganism(s) of concern to grow, survive or be inactivated in raw materials and
intermediate or end products under reasonably foreseeable food processes, storage and use conditions.
The objective and the scope of the document are to determine the experimental design and the selection
of the study conditions. Regulatory authorities can have different recommendations, and these
differences have been included as much as possible. It is, however, possible that specific requirements
should be incorporated to get regulatory approval of the challenge test.
As growth and inactivation kinetics are clearly different, the ISO 20976 series consists of two parts,
under the general title, Microbiology of the food chain — Requirements and guidelines for conducting
challenge tests of food and feed products:
— Part 1: Challenge tests to study growth potential, lag time and maximum growth rate
— Part 2: Challenge tests to study inactivation potential and kinetics parameters (to be developed)
The use of the ISO 20976 series involves expertise in relevant areas, such as food microbiology, food
science, food processing and statistics. The statistical expertise encompasses an understanding of
sampling theory and design of experiments, statistical analysis of microbiological data and overview
of scientifically recognized and available mathematical concepts used in predictive modelling. Even
though many mathematical models are available to describe and predict bacterial growth, the gamma-
[22]
concept (γ-concept) is particularly useful for further simulations using the data generated from
the challenge test, e.g. to assess the growth at storage temperatures other than the one(s) tested, or in
helping to design better food formulations and storage conditions, and thus improving the microbial
quality and/or safety of the food under consideration.
For practical reasons, the term “food” includes feed.
vi © ISO 2019 – All rights reserved

INTERNATIONAL STANDARD ISO 20976-1:2019(E)
Microbiology of the food chain — Requirements and
guidelines for conducting challenge tests of food and feed
products —
Part 1:
Challenge tests to study growth potential, lag time and
maximum growth rate
1 Scope
This document specifies protocols for conducting microbiological challenge tests for growth studies on
vegetative and spore-forming bacteria in raw materials and intermediate or end products.
The use of this document can be extended to yeasts that do not form mycelium.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
ISO 18787:2017, Foodstuffs — Determination of water activity
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
bacterial spore
resistant form of bacteria that is dormant until the germination (3.9) step
3.2
batch
group or set of identifiable food obtained through a given process under practically identical
circumstances and produced in a given place within one defined production period
Note 1 to entry: The batch is determined by parameters established beforehand by the organization and may be
described by other terms, e.g. lot.
[SOURCE: Commission Regulation (EC) No 2073/2005]
ISO 20976-1:2019(E)
3.3
cardinal value
estimated minimal, optimal and maximal values of physico-chemical factors (e.g. temperature, pH, a )
w
that characterize the growth of a given microbial strain
3.4
control unit
unit of food identical to the test unit (3.24) but not artificially contaminated (used as a blank)
3.5
challenge test
study of the growth or inactivation of microorganism(s) artificially inoculated in food
3.6
experimental datapoint
result of analysis of a test unit (3.24) per unit weight (log cfu/g), per unit volume (log cfu/ml), or per
10 10
unit area (log cfu/cm )
Note 1 to entry: For specific cases, the enumeration results may be expressed in log MPN.
3.7
exponential growth phase
phase in which the microbial population is exponentially multiplying as rapidly as possible; growth is
dependent on the growth medium and environment (temperature, humidity, etc.)
Note 1 to entry: Figure 1 describes the three phases of microbial growth kinetics.
3.8
generation time
Tg
time it takes for the microorganisms to increase by a factor 2, also known as doubling time
3.9
germination
mechanism in which a bacterial spore (3.1) starts becoming a vegetative cell (3.25)
3.10
growth potential
Δ
difference between the decimal logarithm of the highest concentration of the target microbial population
(log ) and the decimal logarithm of the initial concentration of this microbial population (log )
max i
Note 1 to entry: The log and log refer to concentrations and are expressed in log cfu/g or log cfu/ml or
max i 10 10
log cfu/cm
3.11
maximum growth rate
kinetics parameter to characterize the exponential growth phase (3.7), represented by the slope of
the curve showing the evolution of the natural logarithm (μ ) or decimal logarithm (V ) of the
max max
population as a function of time, under constant growth conditions
3.12
inoculum
microbial suspension at the desired concentration used to contaminate test units (3.24)
3.13
lag phase
phase, directly after inoculation, during which the microbial population is adapting to the environment,
before it enters the exponential growth phase (3.7)
Note 1 to entry: Figure 1 describes the three phases of microbial growth kinetics.
2 © ISO 2019 – All rights reserved

ISO 20976-1:2019(E)
3.14
lag time
λ
kinetics parameter in time unit to characterize the lag phase (3.13)
3.15
pH value
measure of the concentration of acidity or alkalinity of a material in an aqueous solution
[SOURCE: ISO 5127:2017, 3.12.2.29, modified — Notes 1 and 2 to entry have been removed.]
3.16
primary model
mathematical model describing the changes of microbial counts as a function of time
3.17
organizing laboratory
laboratory with responsibility for managing the challenge tests (3.5)
3.18
sampling
selection of one or more units or portions of food such that the units or portions selected are
representative of that food
3.19
sampling point
time at which the test units (3.24) are analysed and which are represented as experimental datapoints
(3.6) on the kinetics graph
3.20
secondary model
mathematical model describing the effects of the environmental factors (e.g. temperature, pH, a ) on
w
the parameters of the primary model (3.16) (e.g. growth rate)
3.21
sporulation
mechanism by which vegetative cell (3.25) forms spore
3.22
stationary phase
phase in which the microbial population is at its maximum level
Note 1 to entry: Figure 1 describes the three phases of microbial growth kinetics.
3.23
test portion
measured (volume or mass) representative sample taken from the test unit (3.24) for use in the analysis
[SOURCE: ISO 6887-1:2017, 3.5, modified — The end of the definition has been changed from “taken
from the laboratory sample for use in the preparation of the initial suspension” and the Note 1 to entry
has been removed.]
3.24
test unit
measured (volume or mass) amount of the food used for inoculation
3.25
vegetative cell
state of microbial form that is capable of growing under favourable environmental conditions
ISO 20976-1:2019(E)
3.26
water activity
a
w
ratio of the water-vapour pressure in the foodstuff to the vapour pressure of pure water at the same
temperature
[SOURCE: ISO 18787:2017, 3.1, modified — The definition has been condensed and the formula and
Notes 1 and 2 to entry have been removed.]
4 Principle
4.1 General
The aim of the study shall be clearly defined (e.g. assessment/validation of the food shelf-life as a control
measure, assessment of microbial stability). The experimental design shall be in accordance with that
purpose and shall take into account the steps of the food chain for which microbial growth is assessed.
The decision criteria shall be clearly defined (see 7.2).
Knowledge from the FBO on its products (e.g. characteristics or production process) shall be combined
with expertise in food microbiology and analytical sciences to ensure the robustness of the study. The
organizing laboratory shall have knowledge and skills in food microbiology, food science and technology,
and statistics to design and conduct the studies, interpret the results and draw the conclusions. The
analyses shall be conducted under a quality assurance system (e.g. in accordance with ISO/IEC 17025).
Challenge tests aim at studying the growth potential or growth kinetics (lag time and maximum
growth rate) in order to assess, for example, the food shelf-life as a control measure or the microbial
stability of a food.
Growth potential studies are most appropriate to:
— validate the microbiological shelf-life of a food under reasonably foreseeable conditions of use and
storage between production and consumption, ensuring relevant microbiological criteria are met
throughout the product shelf-life;
— assess if a product, tested under specific conditions, supports the growth of the inoculated
microorganism.
Such challenge tests will only validate the specific food characteristics and conditions applied for the
study. When microbiological criteria are not fulfilled or conditions (e.g. food formulation, physico-
chemical characteristics, type and/or concentration of preservatives added, packaging, storage
temperature) are changed, a new growth potential study needs to be carried out in order to validate
the new conditions.
Growth kinetics studies are most appropriate for:
— assessing the effect(s) of intrinsic (e.g. pH, a , preservatives) and extrinsic characteristics (e.g.
w
gas composition, temperature) that have a significant impact on the behaviour of the target
microorganism;
— providing data for developed models to simulate the effect of such factors on microbial behaviour in
the studied food under reasonably foreseeable storage conditions (time and temperature);
— comparing the simulation results to ensure that relevant microbiological criteria are met throughout
the food shelf-life.
Growth kinetics studies are used to estimate and validate the microbiological shelf-life of a food.
They are particularly suitable in the last steps of the food development, including reformulation, new
packaging, and alternative processing conditions.
4 © ISO 2019 – All rights reserved

ISO 20976-1:2019(E)
A growth kinetics study can be more informative than a growth potential study. However, growth
kinetics studies are more complex in terms of study design, execution, results interpretation and
exploitation, particularly in cases where various factors are included.
The behaviour of a microbial population in a food, i.e. microbial growth kinetics, is dependent on the
characteristics of the food (e.g. a , pH, preservatives concentrations), the food storage conditions
w
(temperature, packaging format and gas composition), the food processes, the physiological state of the
microorganism and interactions with the natural background microorganisms.
Microbial growth kinetics are defined by three major phases (see Figure 1).
a) Lag phase: This phase is characterized by the lag time (λ), which corresponds to the intersection
between the exponential growth phase line (plotted in semi-log coordinates) and the horizontal
[15][18]
line crossing through the initial cell concentration . For spore-forming microorganisms, lag
time includes spore germination and outgrowth.
Lag time is dependent on the food characteristics (e.g. physico-chemical and microbiological),
inoculation levels and storage conditions (e.g. temperature, relative humidity, gas composition).
Lag time is also dependent on the physiological state of the microorganism contaminating the food
and any stress experienced by these cells or spores.
b) Exponential growth phase: This phase is characterized by the growth rate (µ or V ), which
max max
corresponds to the maximum increase in natural or decimal logarithm of cell number per unit of time.
The growth rate corresponds to the slope of the curve showing the evolution of the natural
logarithm (µ ) or decimal logarithm (V ) of the population over time during the exponential
max max
phase. The food characteristics (e.g. physico-chemical and microbiological) and storage conditions
(e.g. temperature, relative humidity, gas composition) can significantly influence microbial growth
rates. The growth rate of a microbial population is unaffected by its initial concentration and
physiological states.
The relationship between the generation time (Tg) and µ is given by Formula (1):
max
μ =ln 2 /Tg (1)
()
max
The slope of the curve plotting the log of the microbial population against time, V , and its
10 max
relationship to maximum growth rate is given by Formula (2):
μ =⋅V ln 10 (2)
()
maxmax
c) Stationary phase: In this phase, the microbial population is at its maximum level.
ISO 20976-1:2019(E)
Key
Y log (cfu/g)
X time (days)
1 lag phase
2 exponential growth phase
3 stationary phase
Figure 1 — Microbial growth kinetics with three major phases
4.2 Estimation of the growth potential
The food characteristics (e.g. physico-chemical and microbiological) and storage conditions (e.g.
temperature, relative humidity, gas composition) can significantly influence the microbial growth
potential.
The inoculum shall be adapted to conditions that mimic the microbial cell or spore injury induced by
food handling/processing or any phenomena that can trigger subsequent adaptive responses to growth
conditions, in order to mimic natural microbial behaviour in the food.
This type of test is designed to estimate the changes in concentration of the microbial population during
the challenge test. These tests can be used to determine whether there is significant microbial growth
in a foodstuff and to quantify the increase in the microbial population under a given set of storage
conditions.
It is important to have a minimum of five sampling points that are evenly distributed across the entire
shelf-life, to get an accurate estimation of the growth potential (see 14.2).
Growth potential does not provide information on the length of the lag phase, growth rate value or
maximum stationary-phase level. This makes the growth potential unsuited for extrapolating the
results to other conditions.
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ISO 20976-1:2019(E)
The growth potential can be obtained using dynamic temperature profiles applied to the food during
the study mimicking the food storage conditions.
4.3 Estimation of the growth kinetics parameters (lag time and maximum growth rate)
The growth kinetics characterization consists of the estimation of the lag time and maximum
growth rate. The maximum growth rate is mainly used in assessing, determining and optimizing the
microbiological shelf-life of the food.
Maximum growth rates can be used to directly calculate an increase in microbial counts under the
conditions of the challenge test and/or as inputs for growth simulation.
The experimental design shall generate at least eight experimental data points distributed across all
growth phases, with a minimum of five data points in the exponential phase. Growth kinetics shall
be estimated at a constant temperature by fitting a recognized and commonly accepted mathematical
model used for describing microbial growth.
5 Apparatus
Routine microbiology labware specified in ISO 7218 is required. Specific labware may also be needed to
prepare the test portions, to store them under suitable conditions or monitor how their characteristics
change during the challenge test study. These include the following.
5.1 Apparatus for packaging, the samples under air, under vacuum or under a protective modified
atmosphere.
5.2 Chilled incubator, able to reach and hold setpoint temperatures to ±1 °C.
5.3 Climate-control chamber, able to reach and hold setpoint temperatures to ±1 °C and to adjust
relative humidity to ±10 %.
5.4 pH meter, able to perform measurements to a tolerance of ±0,1 pH units. pH meters shall give
readings to a resolution of 0,01 pH units.
5.5 a meter, meeting the requirements of the ISO 18787.
w
5.6 Headspace gas analyser, to measure gas composition (e.g. O , CO ).
2 2
5.7 Logger for measuring temperature storage conditions of the test unit.
5.8 Logger for measuring relative humidity storage conditions of the test unit.
6 Culture media and reagents
Follow current laboratory practices as specified in ISO 7218.
For the preparation and performance testing of culture media and reagents, follow the procedures as
specified in ISO 11133 and in the International Standard specific to the microbial population studied. Use
internationally recognized and widely accepted methods or alternative methods validated according
to internationally accepted protocols for the detection or enumeration of target microorganisms (e.g.
ISO 16140-2).
ISO 20976-1:2019(E)
7 Study design and sampling
7.1 General
The study design shall address sources of variability, including:
— the inter-batch variability;
— the intra-batch variability;
— the variability in the artificial inoculation of the test units.
7.2 Setting decision criteria for growth potential
Depending on the aim of the challenge test, decision criteria shall be defined before the start of the
study. A two-step approach is used for growth potential study: one step to determine if growth occurs
and the second to determine how much growth is acceptable.
For example, for Listeria monocytogenes, some institutions consider that a food supports growth when
[10][12]
the growth potential is greater than a cut-off value of 0,5 log , whereas others set this cut-off
[13]
value at 1 log .
7.3 Number of batches and selection criteria
The number of batches to be included in the study depends on the variability of the food production
process and food, especially in regard to the intrinsic (e.g. pH, a , preservatives) and extrinsic (e.g.
w
gas composition) characteristics and microbiological properties of the food. This variability shall be
documented. The characteristics of the studied batches shall be representative of the variability of the
production process based on historical data.
If inter-batch variability of food characteristics (e.g. physico-chemical and microbiological) is
sufficient to induce differences in microbial growth behaviour (when either Δφ or Δψ is over
pHaw
0,2), it is necessary to study different batches in order to evaluate the variability within the microbial
[14]
responses . In that case, a minimum number of three batches should be used for both growth
potential and the growth kinetics studies.
The use of a single batch shall be clearly justified, for example:
a) evaluating the impact of a new formulation of the food;
b) using a batch representing the most favourable growth conditions (worst case);
c) applicable for a growth kinetics study only if the impact of the inter-batch variability determined
by the calculator tool (see Annex A) is not significant.
Annex A provides an appropriate calculation scheme for assessing the impact of the inter-batch
variability of the food, pH and a on the behaviour of the microorganism under test. Annex A is only
w
applicable in cases where the challenge tests are conducted to estimate growth rate and where the pH
and a are the only relevant food characteristics having a significant impact on this kinetics parameter.
w
7.4 Preparation of the test units
Test units representative of the food matrix can be either:
— the complete content of the packaging units,
— aseptically-sampled portions from the packaging unit(s) or from the bulk food.
The tests units shall be maintained at an appropriate storage temperature before inoculation. The test
units shall be inoculated as close as possible to the day of production unless otherwise defined by the
8 © ISO 2019 – All rights reserved

ISO 20976-1:2019(E)
objectives of the study. The test units shall have the same composition as the original food, especially
for composite food.
The test units shall be packed using the same packaging conditions as the original food over the shelf-life
under test. It is recommended to use the same packaging material, comparable gas mix and headspace-
food ratio. If, for example, for practical reasons, it is not possible to use the original food packaging,
or packaging material, the use of alternative packaging material shall be justified. The alternative
packaging material shall have the same technical properties (e.g. gas permeability) as the original.
Prepare the number of units to be used for the inoculation of microorganisms (test units) and for the
control (control units, see Clauses 10 and 11). It is recommended that additional test units are prepared
to cover unforeseen incidents.
7.5 Number of test units to be inoculated
The minimum number of test units to be inoculated for analysis per sampling point will depend on the
inter-batch variability (see Annex B). Depending on the intra-batch variability, the number of test units
and/or sampling point may have to be increased.
The sources of variability in artificial inoculations include the type and structure of the food matrix,
as well as the inoculation procedure. When the three batches are not simultaneously inoculated, the
number of test units to be prepared for Time 0 (t ) should be at least three per batch.
Annex B presents a flowchart for determining the minimum number of test units needed for inoculation,
depending on the expected inter-batch variability, the type of challenge test and the minimum number
of sampling points.
NOTE Microbiological growth can be followed by rapid inactivation (depending on the food composition
and storage conditions). The sampling points are divided over the storage period in order to detect a possible
decrease in levels after initial growth.
See Clause 11 for the number of test units to be prepared for the control tests.
8 Selection of strains
Each strain used shall be characterized biochemically and/or serologically and/or genetically in
sufficient detail for its identity to be known. Strains previously isolated from the food matrix (raw
materials, ingredients, end products) or from the production environment or from clinical/food/
environmental samples in outbreaks involving the food are preferred compared to strains from a
culture type collection. The original source of all isolates should be known and the isol
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